(a) Field of the Invention
The invention relates to hydrophobic GRF analogs with increased biological potency and prolonged activity, their application as anabolic agents and treatment of growth hormone deficiencies.
(b) Description of Prior Art
Growth hormone (GH) or somatotropin, secreted by the pituitary gland constitutes a family of hormones which biological activity is fundamental for the linear growth of a young organism but also for the maintenance of the integrity at its adult state. GH acts directly or indirectly on the peripheral organs by stimulating the synthesis of growth factors (insulin-like growth factor-I or IGF-I) or of their receptors (epidermal growth factor or EGF). The direct action of GH is of the type referred to as anti-insulinic, which favors the lipolysis at the level of adipose tissues. Through its action on IGF-I (somatomedin C) synthesis and secretion, GH stimulate the growth of the cartilage and the bones (structural growth), the protein synthesis and the cellular proliferation in multiple peripheral organs, including muscles and the skin. Through its biological activity, GH participates within adults in the maintenance of a protein anabolism state, and plays a primary role in the tissue regeneration phenomenon after a trauma.
The decrease of GH secretion with age, demonstrated in humans and animals, favors a metabolic shift towards catabolism which initiates or participates in the aging of an organism. The loss in muscle mass, the accumulation of adipose tissue, the bone demineralization, the loss of tissue regeneration capacity after an injury, which are observed in elderly, correlate with the decrease in the secretion of GH.
GH is thus a physiological anabolic agent absolutely necessary for the linear growth of children and which controls the protein metabolism in adults.
The secretion of GH by the pituitary gland is principally controlled by two hypothalamic peptides, somatostatin and growth hormone releasing factor (GRF). Somatostatin inhibits its secretion, whereas GRF stimulates it.
The human GH has been produced by genetic engineering for about ten years. Until recently most of the uses of GH were concerned with growth delay in children and now the uses of GH in adults are being studied. The pharmacological uses of GH and GRF may be classified in the following three major categories.
Treatments with recombinant human growth hormone have been shown to stimulate growth in children with pituitary dwarfism, renal insufficiencies, Turner""s syndrome and short stature. Recombinant human GH is presently commercialized as an xe2x80x9corphan drugxe2x80x9d in Europe and in the United States for children""s growth retardation caused by a GH deficiency and for children""s renal insufficiencies. The other uses are under clinical trial investigation.
A decrease in GH secretion causes changes in body composition during aging. Preliminary studies of one-year treatment with recombinant human GH reported an increase in the muscle mass and in the thickness of skin, a decrease in fat mass with a slight increase in bone density in a population of aged patients. With respect to osteoporosis, recent studies suggest that recombinant human GH does not increase bone mineralization but it is suggested that it may prevent bone demineralization in post-menopausal women. Further studies are currently underway to demonstrate this theory.
In preclinical and clinical studies, growth hormone has been shown to stimulate protein anabolism in wound and bone healing in cases of bum, AIDS and cancer.
GH and GRF are also intended for veterinary pharmacological uses. Both GH and GRF stimulate growth in pigs during its fattening period by favoring the deposition of muscle tissue instead of adipose tissue and increase milk production in cows, and this without any undesired side effects which would endanger the health of the animals, and without any residue in the meat or milk being produced. The bovine somatotropin (BST) is presently commercialized in the United States.
Most of the clinical studies undertaken were conducted with recombinant GH. GRF is considered as a second generation product destined to replace, in the near future, the use of GH in most instances. Accordingly, the use of GRF presents a number of advantages over the use of GH per se.
cl Physiological Advantages
Growth hormone (GH) is secreted by the pituitary gland in a pulse fashion. Since this rhythm of secretion is crucial for an optimal biological activity, the administration of GH to correspond to its natural mode of secretion is difficult to achieve. When GRF is administered in a continuous fashion as a slow releasing preparation or as an infusion, it increases GH secretion while respecting its pulsatility.
The recombinant GH which is presently commercialized is the 22 kDa form whereas GRF induces the synthesis and secretion from the pituitary gland of all the chemical isomers of GH which participate in a wider range of biological activities.
A treatment with GH results in a decreased capacity of the pituitary gland to secrete endogenous growth hormone, and the GH response to GRF is diminished after such a treatment. On the contrary, a treatment with GRF does not present this disadvantage, its trophic action on the pituitary gland increases this gland""s secreting capacity in normal animals and in patients with somatotroph insufficiency.
The production of GH by genetic engineering is very expensive for clinical use. In particular, there are risks of contamination of these commercial preparations with material from the bacterial strain used. These bacterial contaminants may be pyrogens or may result in immunogenic reactions in patients. The purification of the recombinant product is carried out by following a plurality of successive chromatography steps. The drastic purity criteria imposed by regulatory agencies necessitate multiple quality control steps.
On the other hand, the synthesis of GRF is of chemical nature. The synthesis is carried out in a solid phase and its purification is done in a single step using high performance liquid chromatography (HPLC). Also the quantity of GRF to be administered is much less than the quantity of GH for the same biological result.
Even with all these advantages, GRF is still not commercialized as a therapeutic agent to date, mainly because of its instability. The human GRF is a peptide of 44 amino acids of the following sequence:
The minimum active core is hGRF (1-29)NH2 
As for many peptides, hGRF (1-29)NH2 is rapidly degraded in a serum medium and its metabolites have no residual biological activity. It has been well established that the action of enzymes, namely that of dipeptidylaminopeptidase type IV, in a blood medium results in the hydrolysis of the peptide bond Ala2-Asp3 of GRF. This hydrolysis results in a multitude of negative consequences which were the subject of many studies reported in the literature. Essentially, this hydrolysis leads to the formation of truncated peptides of specific activity reduced to less than 1/1000 of the biological activity.
Clinical studies with children and adults have confirmed that natural hGRF (1-44)NH2 or the active fragment hGRF (1-29)NH2 are not potent enough to produce equal effects corresponding to those of recombinant GH.
It is well known that the anchoring of hydrophobic groups, such as xe2x80x94NEt2 at the C-terminal of a peptidic sequence can result in a significantly increased specific activity. In terms of hydrophobicity, these results are contradicted by a fair number recent works such as those of Muranichi (S. Muranichi et al., 1991, Pharm. Res., 8:649-652) which stress the inefficacy of the lauroyl group as a hydrophobic group at the N-terminal to create small peptide analogs having the desired biological activity. Hence, the contradictory investigations of the prior art failed to address the issue of finding a more potent GRF analog using hydrophobic residues.
Gaudreau et al. (P. Gaudreau et al., 1992, J. Med. Chem., 35(10),: 1864-1869) describe the affinity of acetyl-, 6-aminohexanoyl-, and 8-aminooctanoyl-GRF(1-29)NH2 with the rat pituitary receptor. In this report, none of the fatty acid-GRF compounds tested exhibited a higher affinity than hGRF(1-29)NH2 itself, and the authors concluded that xe2x80x9c. . . modifications to increase the hydrophobic character at the N-terminus of hGRF(1-29)NH2 do not constitute a suitable approach to increase receptor affinity.xe2x80x9d
Coy et al. (D. H. Cow et al., 1987, J. Med. Chem., 30:219-222) describe an acetyl-GRF peptide with an increased biological activity on a rat model, more particularly on a rat anesthetized with sodium pentobarbital. The in vitro GH response by cultured rat pituitary cells was also analyzed. However, these authors did not synthesize and test fatty acid-GRF analogs with a carbon chain longer than two (2) carbon atoms (acetyl group) added at the N-terminus region of the GRF and acetyl cannot be considered a hydrophobic group.
Up to now, most of the GRF analogs described (including those of Gaudreau et al. and those of Coy et al.) have been tested in rat models, either in vitro or in vivo. Since human and rat GRF(1-29)NH2 are markedly different, the structure-activity relationships of GRF are different in both species. Therefore, it is not possible to extrapolate results obtained in rats to humans.
Accordingly, it is necessary to design GRF analogs with improved anabolic potency and having a prolonged activity. This increased potency could result from a resistance to serum degradation and/or from hyperagonistic properties.
It would be highly desirable to be provided with GRF analogs with increased anabolic potency.
One aim of the present invention is to provide new biodegradable GRF analogs with improved biological potency and prolonged activity.
Another aim of the present invention is to provide GRF analogs with increased anabolic potency and prolonged activity, i.e. capable to substantially elevate insulin-like growth factor I (IGF-I) levels when chronically administered in humans and animals.
Another aim of the present invention is to provide a means to render any GRF analog more biologically potent and with a prolonged activity.
Another aim of the present invention is to provide a method of producing active GRF analogs with improved anabolic potency and prolonged activity.
The present invention relates to the preparation of hydrophobic GRF analogs. These chimeric analogs include a hydrophobic moiety (tail), and can be prepared, either by anchoring one or several hydrophobic tails to the GRF, or by substituting one or several amino-acids by a pseudomicellar residue in the chemical synthesis of GRF. The GRF analogs in accordance with the present invention are characterized in that:
a) These analogs possess an enhanced biological activity; specifically, they are able to markedly increase GH and IGF-I blood levels when administered in an animal model closely related to human. This characteristic is particularly advantageous in that it results in a reduced dosage of an hyperactive compound being administered to the patient, thus improving treatment efficacy and reducing treatment costs.
b) Both natural amino acid and hydrophobic substances, such as fatty acids, are used for the chemical synthesis of the GRF analogs.
c) They present a high biological activity at infinitely small dosages.
d) They remain active for a prolonged period of time, with a high biological activity.
The use of fatty bodies in accordance with the present invention results in GRF analogs which overcome all the drawbacks of the prior art.
The GRF analogs of the present invention exhibit improved anabolic potency with a reduced dosage and have a prolonged activity. Furthermore, the present invention deals with GRF and any of its analogs, truncated or substituted.
In accordance with the present invention there is provided a hydrophobic GRF analog of formula A:
Xxe2x80x94GRF-peptidexe2x80x83xe2x80x83(A)
wherein;
the GRF peptide is a peptide of formula B;
A1-A2-Asp-Ala-Ile-Phe-Thr-A8-Ser-Tyr-Arg-Lys-A13-Leu-A15-Gln-Leu-A18-Ala-Arg-Lys-Leu-Leu-A24-A25-Ile-A27-A28-Arg-A30-R0xe2x80x83xe2x80x83(B)
xe2x80x83wherein,
A1 is Tyr or His;
A2 is Val or Ala;
A8 is Asn or Ser;
A13 is Val or Ile;
A15 is Ala or Gly;
A18 is Ser or Tyr;
A24 is Gln or His;
A25 is Asp or Glu;
A27 is Met, Ile or Nle;
A28 is Ser or Asn;
A30 is a bond or any amino acid sequence of 1 up to 15 residues;
R0 is NH2 or NH-(CH2)n-CONH2, with n=1 to 12 and;
X is a hydrophobic tail anchored via an amide bond to the N-terminus of the peptide and said hydrophobic tail defining a backbone of 5 to 7 atoms;
wherein said backbone can be substituted by C1-6 alkyl, C3-6 cycloalkyl, or C6-12 aryl;
and comprises at least one rigidifying moiety connected to at
least two atoms of the backbone;
said moiety selected from the group consisting of double bond, triple bond, saturated or unsaturated
C3-9 cycloalkyl, and C6-12 aryl.
By the term rigidifying moiety is meant a moiety that will confer rigidity to the hydrophobic tail. The rigidifying moiety connects at least two atoms which are part of the backbone of the hydrophobic tail. For example, the backbone of the following hydrophic tail is as follows: 
Preferably, the backbone is substituted with one rigidifying moiety selected from the group consisting of double bond, triple bond, saturated C3-7 cycloalkyl and C6 aryl.
Also preferably, the backbone is substituted with 2 rigidifying moieties which are independently selected from the group consisting of double bond and saturated or unsaturated C3-9 cycloalkyl.
More preferably, the backbone is substituted with 2 rigidifying moieties which are independently selected from the group consisting of double bond, triple bond, saturated C3-7 cycloalkyl and C6 aryl.
In an alternative embodiment, the backbone is substituted one rigidifying moiety selected from the group consisting of double bond, triple bond, saturated C3-7 cycloalkyl and C6 aryl, which are located at the 3,4-positions, the 3,5-positions or the 3,6-positions of the backbone.
Preferably, the hydrophobic tail is selected from the group consisting of: 
In accordance with the present invention, there is provided a method of increasing the level of growth hormone in a patient which comprises administering to said patient an effective amount of a GRF analog of the present invention.
In accordance with the present invention, there is provided a method for the diagnosis of growth hormone deficiencies in patients, which comprises administering to said patient a GRF analog of the present invention and measuring the growth hormone response.
In accordance with the present invention, there is provided a method for the treatment of pituitary dwarfism or growth retardation in a patient, which comprises administering to said patient an effective amount of a GRF analog of the present invention.
In accordance with the present invention, there is provided a method for the treatment of wound or bone healing in a patient, which comprises administering to said patient an effective amount of a GRF analog of the present invention.
In accordance with the present invention, there is provided a method for the treatment of osteoporosis in a patient, which comprises administering to said patient an effective amount of a GRF analog of the present invention.
In accordance with the present invention, there is provided a method for improving protein anabolism (including protein sparing effect) in human or animal, which comprises administering to said human or animal an effective amount of a GRF analog of the present invention.
In accordance with the present invention, there is provided a method for inducing a lipolytic effect in human or animal afflicted with clinical obesity, which comprises administering to said human or animal an effective amount of a GRF analog of the present invention.
In accordance with the present invention, there is provided a method for the overall upgrading of somatotroph function in human or animal, which comprises administering to said human or animal an effective amount of a GRF analog of the present invention.
In the present invention the amino acids are identified by the conventional three-letter abbreviations as indicated below, which are as generally accepted in the peptide art as recommended by the IUPAC-IUB commission in biochemical nomenclature:
The term xe2x80x9cnatural amino acidxe2x80x9d means an amino acid which occurs in nature or which is incorporated as an amino acid residue in a naturally occurring peptide. In addition, the abbreviation Nle is intended to mean Norleucine.
All the peptide sequences set out herein are written according to the generally accepted convention whereby the N-terminal amino acid is on the left and the C-terminal amino acid is on the right.